Hydrogen peroxide sterilization method

ABSTRACT

A method of metering a preselected volume of hydrogen peroxide into a vessel under vacuum is disclosed. The method comprises the steps of connecting a passage of known volume to the vessel under vacuum to evacuate the passage; sealing the passage; connecting the passage to a supply of hydrogen peroxide solution for a time sufficient to draw the hydrogen peroxide solution into the evacuated passage and fill the passage with the hydrogen peroxide solution; sealing the passage; and repeating steps a) to d) until a cumulative volume of fills of the passage is equal to the preselected volume. The volume of the passage is preferably 15 μL to 75 μL. Sterilization is controlled in an economic manner by obviating the need for a means to measure the hydrogen peroxide concentration in the chamber.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/893,742, filed Sep. 29, 2010 and entitled STERILIZATION METHODAND APPARATUS, which claims priority from U.S. Provisional ApplicationSer. No. 61/247,197, filed Sep. 30, 2009 and entitled STERILIZATIONMETHOD AND APPARATUS, the contents of which are incorporated into thepresent application in their entirety.

FIELD OF THE INVENTION

The present disclosure relates generally to sterilization methods andapparatus. More particularly, the present disclosure relates to asterilization process using gaseous biocides under vacuum.

BACKGROUND OF THE INVENTION

Sterilization is the destruction of any virus, bacteria, fungus or othermicro-organism, whether in a vegetative or in a dormant spore state andis defined by a 10⁻⁶ reduction in the level of bacteria. Conventionalsterile processing procedures for medical instruments involve hightemperature (such as steam and dry heat units) or chemicals (such asethylene oxide gas, hydrogen peroxide, or ozone).

Sterilization methods and apparatus using gaseous sterilants are wellknown. Sterilizers using hydrogen peroxide as the sterilant are widelyused. The hydrogen peroxide is generally supplied as an aqueous solutionand evaporated prior to injection into a sterilization chamber of thesterilizer, by heating of the solution, or by applying a vacuum to thesterilization chamber, or both. After evaporation of the solution, thesterilization atmosphere in the sterilization chamber includes watervapor and hydrogen peroxide gas. It is a disadvantage of this processthat the water vapor tends to condensate on articles in the chamber asthe sterilization proceeds. The resulting layer of water condensate onthe articles to be sterilized interferes with the sterilizing action ofthe hydrogen peroxide. Numerous apparatus and process modifications havebeen developed to address this problem, all of which are aimed atlimiting the relative humidity in the sterilization atmosphere duringthe sterilization process. However, these modifications invariablyincrease operating cost and/or sterilization cycle times.

Sterilization processes using both hydrogen peroxide gas and ozone gashave been used, but with unsatisfactory results especially with respectto the sterilization of articles with long internal lumens, such asgastroscopes and colonoscopes, and with respect to cycle times andsterilization cost. Although ozone based processes are satisfactory withrespect to sterilization of articles with long lumens, materialcompatibility represents a problem. Hydrogen peroxide based processesare generally unsatisfactory regarding the sterilization of long lumens.Undesired condensation of the hydrogen peroxide on the article to besterilized reduces the sterilization efficiency.

Therefore, a method and apparatus is desired which would address atleast one of the disadvantages of known sterilization processes usinggaseous sterilants.

SUMMARY OF THE INVENTION

It is an object of the present invention to obviate or mitigate at leastone disadvantage of previous sterilization processes using gaseoussterilants.

In a first aspect, a method is provided for metering hydrogen peroxidegas into an evacuated vessel, for example a sterilization chamber, or anevaporator therefore, for controlling the concentration of hydrogenperoxide in the chamber. The method includes the steps of a) connectinga passage of known volume to the vessel under vacuum to evacuate thepassage; sealing the passage; connecting the passage to a supply ofhydrogen peroxide solution for a time sufficient to draw the hydrogenperoxide solution into the evacuated passage and fill the passage withthe hydrogen peroxide solution; sealing the passage; and repeating stepsa) to d) until a cumulative volume of fills of the passage is equal tothe preselected volume. The concentration of the hydrogen peroxide vaporin the chamber is controlled in a repeatable manner by simply monitoringthe number of injection cycles until the cumulative volume of injectionis achieved.

In a second aspect, the known volume of the metering passage is between75 μL and 15 μL.

In a third aspect, the volume of the metering passage is between 15 μLand 35 μL.

In a fourth aspect, the volume of the metering passage is between 15 μLand 20 μL.

In the method of this disclosure, expensive peroxide concentrationmeasurement systems are replaced with an economical monitoring of thenumber of injection cycles using a passage of fixed volume.

The method includes the step of maintaining the sterilization chamber ata vacuum pressure below the pressure at which hydrogen peroxide willboil at the preselected temperature.

Other aspects and features of the present invention will become apparentto those ordinarily skilled in the art upon review of the followingdescription of specific embodiments of this disclosure in conjunctionwith the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present apparatus, systems and methods will now bedescribed, by way of example only, with reference to the attachedFigures, wherein:

FIG. 1 shows a schematic diagram of an apparatus in accordance with thisdisclosure, the illustrated parts of the apparatus being listed in TableIII;

FIG. 2 shows a schematic diagram of a hydrogen peroxide delivery systemin accordance with this disclosure, the illustrated parts of the systembeing listed in Table III;

FIG. 3 is a flow diagram of a preferred sterilization method inaccordance with this disclosure;

FIG. 4 is a graph illustrating a first exemplary sterilization cycle inaccordance with this disclosure;

FIG. 5 is a graph illustrating a second exemplary sterilization cycle inaccordance with this disclosure;

FIG. 6 is a graph illustrating a third exemplary sterilization cycle inaccordance with this disclosure;

FIG. 7 shows an exemplary embodiment of a hydrogen peroxide supply unitin accordance with this disclosure;

FIG. 8 shows an exemplary embodiment of a hydrogen peroxide reservoir,metering and evaporation assembly in accordance with this disclosure;

FIGS. 9a, 9b, and 9c , also referenced collectively herein as FIG. 9, isa schematic diagram of a control system for an apparatus in accordancewith this disclosure;

FIG. 10a is a perspective view of a sterilant container in accordancewith this disclosure;

FIG. 10b is a cross-sectional view of the container of FIG, 10 a;

FIG. 10c is a side elevational view of the container of FIG. 10a ; and

FIG. 10d is enlarged detail B of the container shown in FIG. 10 b.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Generally, the present application provides a method for sterilizationof an article in a gaseous sterilization atmosphere by sequentiallyadding evaporated hydrogen peroxide and particularly to a method ofmetering hydrogen peroxide vapor into an evacuated chamber. Preferably,the metering is controlled by using a passage of known volume formetering the amount of hydrogen peroxide injected into the chamber.Moreover, by controlling not only the overall concentration of thehydrogen peroxide in the chamber, but also the individual aliquotsinjected, condensation of hydrogen peroxide in the sterilization chamberat a preselected temperature can be controlled.

The method includes the steps of connecting a passage of known volume tothe evacuated chamber for evacuating the passage; sealing the passage;connecting the evacuated passage to a supply of hydrogen peroxidesolution for a time sufficient to draw the hydrogen peroxide solutioninto and fill the passage with the hydrogen peroxide solution; sealingthe passage; and repeating those steps until a cumulative volume offills of the passage is equal to the preselected volume. Theconcentration of the hydrogen peroxide vapor in the chamber iscontrolled in a repeatable manner by simply monitoring the number ofinjection cycles until the cumulative volume of injection is achieved.

By maintaining the sterilization chamber at a vacuum pressure below thepressure at which hydrogen peroxide will boil at the preselectedtemperature and evaporating and injecting successive pulses of hydrogenperoxide until a desired overall injection volume is achieved, theconcentration of hydrogen peroxide in the chamber can be controlled aswell as unwanted condensation of the hydrogen peroxide at thepreselected temperature.

As will be discussed further below, hydrogen peroxide solution injectedinto the sterilization chamber in a vapor form will condensate on thearticle to be sterilized. However, condensation of the hydrogen peroxideinterferes with the sterilization of long lumens, since the hydrogenperoxide is removed from the vapor phase during condensation. Thus, inorder for the hydrogen peroxide to penetrate long lumens, the hydrogenperoxide should be maintained in the vapor phase as long as possible andcondensation avoided during hydrogen peroxide injection. This is achievein accordance with the present disclosure by controlling the volume ofthe individual hydrogen peroxide injection pulses. In one embodiment,the volume of each pulse of hydrogen peroxide is less than 75 μL. Inother embodiment, the volume of each pulse of hydrogen peroxide is lessthan 35 μL. In a further embodiment, the volume of each pulse ofhydrogen peroxide is less than 20 μL.

This method of controlling hydrogen peroxide concentration can be usedin a sterilization method as illustrated in the flow diagram of FIG. 3and the cycle graphs of FIGS. 4 to 6, wherein an article is sterilizedby exposing it sequentially to hydrogen peroxide and ozone. The articleis preferably exposed under vacuum first to an evaporated aqueoussolution of hydrogen peroxide and subsequently to an ozone containinggas. The exposure to the evaporated hydrogen peroxide is carried out bycontrolling unwanted condensation of hydrogen peroxide. Preferably, theexposure is carried out without reducing the water vapor content of thesterilization atmosphere, the water vapor content being derived from theaqueous solvent of the hydrogen peroxide solution and from thedecomposition of the hydrogen peroxide into water and oxygen. Mostpreferably, the complete sterilization process is achieved while thechamber remains sealed and without removal of any component of thesterilization atmosphere. For this purpose, the chamber is initiallyevacuated to a first vacuum pressure sufficient to cause evaporation ofthe aqueous hydrogen peroxide at the temperature of the chamberatmosphere. The chamber is then sealed and hydrogen peroxide and ozonecontaining gas are sequentially added to the chamber and maintained inthe chamber for a preselected exposure time. All removal of anycomponents in the sterilization atmosphere is stopped during addition ofthe sterilants and for the duration of the exposure time.

The aqueous hydrogen peroxide solution is evaporated and directlyinjected into the sterilization chamber without any measures to reducethe water vapor content. The inventors of the present application havesurprisingly discovered that the amount of sterilants used and thelength of the sterilization cycle can be significantly reduced, when anyand all steps to reduce the water vapor content in the chamber areomitted and the hydrogen peroxide sterilization step is followed by anozone sterilization step, since the water vapor generated during thehydrogen peroxide sterilization step can be used to sufficientlyhumidify the atmosphere in the chamber to improve the ozonesterilization step. Much lower amounts of hydrogen peroxide and ozonecan be used than in prior art processes using the same sterilants, whilestill achieving complete sterilization. Also, the required amounts ofthe sterilants in accordance with the present disclosure are lower thanwhat would be expected from simply using the two sterilants in the samecycle. Thus, maintaining the chamber sealed throughout all sterilizationsteps without any measures to control the humidity in the sterilizationatmosphere appears to result in a synergistic effect.

A sterilizer in accordance with this disclosure as illustratedschematically in FIG. 1 operates generally in the following manner. Anarticle to be sterilized (not shown) is placed into a sterilizationchamber 10 and the chamber is sealed. A vacuum is applied to the chamber10. Evaporated hydrogen peroxide solution is supplied into thesterilization chamber 10 from a delivery unit 30 (see FIG. 8), whichwill be discussed in more detail below. The evaporated hydrogen peroxidesupplied into the chamber provides a partial sterilization of thearticle. Medical quality oxygen is subjected in an ozone generator 22 toan electrical field, which converts the oxygen into ozone containinggas. The ozone containing gas is then fed into the chamber 10, which hasbeen humidified by the injection of the evaporated hydrogen peroxidesolution and the decomposition of the hydrogen peroxide into freeradicals (hydroxyls), water and oxygen. The ozone containing gasfinishes the sterilization of the article. Remaining sterilant gases aresubsequently decomposed into water and oxygen using a catalyst 52. Theonly residues left at the end of the sterilization cycle are oxygen andclean water.

The ozone sterilization method of this disclosure is preferably carriedout at room temperature and, thus, requires substantially no aeration orcooling down of sterilized articles so that they can be used immediatelyfollowing the sterilization cycle. Moreover, the gases used diffuse morequickly into long lumens to be sterilized, reducing the cycle timesrequired for sterilization. This allows hospitals to reduce the cost ofmaintaining expensive medical device inventories. The sterilizationmethod of the invention offers several further advantages. It producesno toxic waste, does not require the handling of dangerous gascylinders, and poses no threat to the environment or the user's health.Stainless-steel instruments and heat-sensitive instruments can betreated simultaneously, which for some users will obviate the need fortwo separate sterilizers.

The preferred sterilization apparatus in accordance with this disclosureas illustrated schematically in FIG. 1 includes a sterilization chamber10 which can be sealed to contain a vacuum. This is achieved with anaccess door 12, which can be selectively opened for access into thechamber and which seals the chamber in the closed condition. Theapparatus further includes an ozone generator 22 for supplyingozone-containing gas to the sterilization chamber, a hydrogen peroxidedelivery unit 30 for supplying evaporated hydrogen peroxide to thesterilization chamber 10, and a vacuum pump 40 (CM-005-052 TSO3, Inc.).The vacuum pump 40 is used for the application of a sufficient vacuum tothe sterilization chamber 10 to increase the penetration of thesterilizing gas and to be able to generate evaporated hydrogen peroxidesolution at a temperature below the temperature inside the sterilizationchamber. The vacuum pump 40 in the preferred embodiment is capable ofproducing a sufficient vacuum in the sterilization chamber to lower theboiling point of water in the chamber below the actual temperature ofthe atmosphere in the chamber. In the preferred apparatus, the vacuumpump is capable of producing a vacuum of 1 Torr (1.33 mbar). Ozoneproduced in the ozone generator 22 is destroyed in an ozone catalyst 52to which ozone-containing gas is fed either after passage through thesterilization chamber 10 or directly from the ozone generator 22 throughby-pass valve 29 b. The ozone catalyst 52 (AM-004-001, TSO₃ Inc.) isconnected in series after the vacuum pump 40 to prevent ozone gasescaping to ambient. The ozone decomposing material in the preferredcatalyst 52 is carulite. For economic and practical reasons, it ispreferred to use a catalyst for decomposition of the ozone in thesterilization gas exhausted from the sterilization chamber 10. Thecatalyst destroys hydrogen peroxide and ozone on contact andretransforms it into oxygen and water with a certain amount of heatbeing produced. Catalysts of this type and their manufacture are wellknown to the person skilled in the art of ozone generators and need notbe described in detail herein. Furthermore, other means for destroyingthe ozone and hydrogen peroxide contained in the sterilization gas willbe readily apparent to a person skilled in the art. For example, the gascan be heated for a preselected time to a temperature at which thesterilant decomposition is accelerated, for example, to 300° C. for aperiod of 3 seconds.

The hydrogen peroxide delivery unit 30 includes a reservoir 220(AM-213-010, TSO₃ Inc.), a metering unit 240, and an evaporator unit 260(FM-213-003, TSO₃ Inc.) directly connected to the sterilization chamber10 through a conduit 280. (AM-213-003, TSO₃ Inc.) The reservoir 220 isequipped with a level sensor 222 to always ensure a sufficiently highlevel of hydrogen peroxide for the execution of another sterilizationcycle. A hydrogen peroxide solution (3-59%) is supplied to the reservoirfrom a hydrogen peroxide supply unit 200 (see FIG. 7), which will bediscussed in more detail below. The hydrogen peroxide solution issupplied into the supply unit 200 from a sealed bottle 180 (see FIG. 7).The evaporated hydrogen peroxide solution produced in the evaporatorunit 260 directly enters the sterilization chamber 10 with nointermediate flow restriction or valve. The evaporator unit ispreferably equipped with a heating device (not shown) that maintains thetemperature of the hydrogen peroxide solution sufficiently high toachieve a higher evaporation rate and prevent freezing of the solution.

The ozone generator 22 (OZ, model 14 a, TSO₃ Inc.) is of the coronadischarge type and is cooled to decrease the ozone decomposition rate,all of which is well known in the art. The ozone generation isassociated with energy loss in the form of heat. Since heat acceleratesthe decomposition of ozone into oxygen, it should be removed as quicklyas possible by cooling of the ozone generator 22. The ozone generator inthe apparatus is kept at the relatively low temperature of 3 to 6° C. bya cooling system 60, which is either an indirect cooling system withcooling water recirculation, or a direct cooling system with an aircooling unit or a refrigeration unit for cooling (not illustrated). Thecooling system is preferably kept at the temperature of 3 to 6° C. Inthe preferred embodiment, the cooling system is kept at 4° C. so thatthe ozone-containing gas generated by generator 22 is at the ambienttemperature of around 20 to 35° C. Thus, the ozone-containing gasentering into the sterilization chamber for humidification andsterilization is kept at ambient temperatures of 20 to 35° C. This meansthat ozone decomposition is minimized and the sterilization process ismost efficient. The ozone-generator 22 is preferably supplied withmedical grade oxygen. Oxygen may also be supplied directly to thesterilization chamber 10 through oxygen supply valve 21. The apparatuscan be connected to a wall oxygen outlet common in hospitals or to anoxygen cylinder or to any other source capable of supplying the requiredquality and flow. The supply of oxygen to the generator 22 takes placeacross a filter 23, a pressure regulator 24, a flow meter 25 and anoxygen shut off valve 26. The generator is protected against oxygen overpressure by a safety pressure switch 27. The ozone-oxygen mixturegenerated by the generator 22 is directed to the sterilization chamber10 through a flow regulator orifice 28 and a mixture supply solenoidvalve 29 a. The mixture can also be directly supplied to the ozonecatalyst 52 by way of a bypass solenoid valve 29 b (optional). In apreferred embodiment in which a sterilization chamber of 125 litersvolume is used, the pressure regulator 24 and the regulator valve 28preferably control the oxygen input at a pressure of about 13.8 kPa (2psig) and a flow rate of about 1.5 litres per minute. However, it willbe readily apparent to the skilled person that other flow rates may beused depending on the make and model of the ozone generator 22 and thesize of the sterilization chamber.

The vacuum in the sterilization chamber 10 is produced by way of thevacuum pump 40 and the sterilization chamber drainage valve 44.

Valves 29 a and 29 b are Teflon solenoid valves (CM-900-156, TSO3 Inc.)Valve 26 and vacuum valve 44 are solenoid valves (CM-015-004, TSO3Inc.).

The preferred ozone generator used in the process and apparatus of thisdisclosure is a generator of the corona discharge type, which is wellknown to the person skilled in the art and need not be further describedherein.

Operation

A preferred sterilization method according to this disclosure includesthe following general steps as illustrated by the flow chart of FIG. 3.Articles to be sterilized, such as medical instruments, can be placeddirectly into the sterilization chamber, but are preferably sealed insterile packaging containers, sterile wraps or pouches such as generallyused in the hospital environment and then placed into the sterilizationchamber. Various different types of such containers or pouches are wellknown to the person skilled in the art and need not be further describedherein.

After insertion of the article to be sterilized has been placed into thesterilization chamber in step 320, the door of the sterilization chamberis closed and the chamber sealed in step 340 and a vacuum is applied tothe sterilization chamber in step 350 until a first pressure of 1 Torr(1.33 mbar) is reached in the chamber. The sterilization chamber wallshave preferably been preheated in a warm-up step 310 to a temperature of40° C. Evaporated hydrogen peroxide solution is admitted into thesterilization chamber in humidification step 360 to partially sterilizeand humidify the chamber contents. The injection of evaporated hydrogenperoxide solution is stopped once a pressure increase of 19 Torr hasbeen achieved in the chamber. The chamber can be maintained sealed for afirst exposure period 370 (preferably 2 minutes) during which thehydrogen peroxide at least partially decomposes into free radicals,water and oxygen. Preferably, this exposure period can also be omitted.An ozone containing gas, preferably in the form of a mixture of dryozone and oxygen is then supplied to the chamber in the ozone injectionstep 380 and the chamber maintained sealed for a preselected secondexposure period 390. No humidification of the ozone containing gas iscarried out, or is even necessary, since the chamber atmosphere has beenhumidified by the hydrogen peroxide solution. Between the application ofthe vacuum, before the hydrogen peroxide evaporation step, and the endof the second exposure period, all removal of any sterilizationatmosphere components is interrupted so that none of the components ofthe atmosphere are removed before the end of the second exposure period.The steps of vacuum application, hydrogen peroxide injection with firstexposure period and ozone gas injection with second exposure period, arepreferably repeated at least once, the number of repetitions beingdetermined in step 395 on the basis of the cycle chosen previously instep 330. To remove all remaining sterilants from the sterilizationchamber 10 after the sterilization cycle is completed a ventilationphase 400 is commenced, which preferably includes multiple cycles ofevacuation of the chamber and flushing with oxygen. After theventilation phase 400, the door is unlocked in step 410 and thesterilized articles can be taken from the chamber. The temperature ofthe floor and door of the chamber and of the evaporator unit ispreferably controlled throughout the sterilization process.

In an exemplary sterilization apparatus in accordance with thisdisclosure, the user has the choice of multiple different sterilizationcycles. In a preferred method, the user can choose in cycle selectionstep 330 of the process among three cycles which have the respectivecharacteristics shown in Table 1 and discussed below.

TABLE I Cycle phases Cycle 1 Cycle 2 Cycle 3 Vacuum 1 Torr 1 Torr 1 TorrHumidification with 50% H202 20 Torr 20 Torr 20 Torr solutionHumidification plateau (optional) 2 min 2 min 2 min 03 Injection 2 mg/110 mg/L 3 mg/L Exposure 5 min 5 min 10 min No of repetition(s) 2 2 4Approx. Cycle duration 46 min 56 min 100 min Cycle 1- Surfacesterilization of devices having low compatibility with ozone, hingeddevices and short flexible endoscopes (1 mm × 85 cm). (Ex. Cameras,cables, paddles, forceps, bronchoscopes, ureteroscopes). Cycle 2-Surface devices with high compatibility with ozone, hinged instrumentsand rigid endoscopes (1 mm × 50 cm). Cycle 3- Instruments sterilizablewith cycle #1 and complex endoscopes (Ex. gastroscopes, colonoscopes).

Although it is preferred to operate the present sterilization processusing a 50% hydrogen peroxide solution, the process can be operated withsolutions including 3% -50% hydrogen peroxide. Exemplary conditions forthe process when operated with a 3%, 30% and 50% hydrogen peroxidesolution are as follows.

TABLE II Max Injection Ozone Pressure dose No of Conditioning % H₂O₂(Torr) (mg/L) repetitions time 3 44-54 25-50  2-8 2 hrs 30 30-44 5-252-6 2 hrs 50 17-21 (20) 2-10 2-4 0 hr 

The maximum injection pressure is the pressure at which injection of theevaporated hydrogen peroxide solution is stopped. The conditioning timerepresents a time period after sealing of the chamber and prior toapplication of the vacuum in which the articles to be sterilized aremaintained in the sterilization chamber and gradually warm up from roomtemperature due to the chamber walls, floor and door being heated toabout 40° C. This warming up of the load in the chamber is required toprevent undue condensation of water on the load on injection of theevaporated hydrogen peroxide solution. The risk of condensationincreases with decreasing hydrogen peroxide solution concentrations.

Once the user has chosen one of the three cycles, the user closes thesterilization chamber door and pushes the start button. The sterilizercontrol system (see FIG. 9) will then, under the control of a built inoperating software, start the sterilization process according to thecycle chosen and using preselected parameters for the cycle chosen.There is no pre-conditioning of the sterilization load. The cycle startswith the generation a vacuum in the sterilization chamber ofapproximately 1 Torr (1.33 mbar). An evaporated aqueous hydrogenperoxide solution is subsequently injected into the chamber through theevaporator unit to partially sterilize and humidify the load. Beforeentering the evaporator unit, the hydrogen peroxide solution passesthrough the metering unit 240 shown in FIG. 8. The metering unit 240 isdirectly connected to the evaporator unit 260 and, thus, subjected tothe vacuum pressure present in the chamber. The metering unit 240includes a base block 241 having a passage of a fixed, known volume (notshown) and connected by an intake valve 242 at an upstream end of thepassage to the hydrogen peroxide reservoir 220 and by an exhaust valve243 at a downstream end of the passage to the evaporator unit 260. Theflow of hydrogen peroxide solution through the metering unit 240 can beexactly controlled by way of the valves 242, 243, which are switchedoppositely and non-overlapping so that one valve is always closed whenthe other is open and both valves are never open at the same time. Inthis manner, the passage is evacuated when the exhaust valve 243 is openand the intake valve 242 is closed, filled with hydrogen peroxidesolution when the exhaust valve 243 is closed and the intake valve 242is open and evacuated again when the exhaust valve 243 is again open andthe intake valve 242 is again closed. Since the exact volume of thepassage is known, the amount of hydrogen peroxide solution supplied pervalve cycle is known and the total amount of hydrogen peroxide can becalculated on the basis of the number of valve switching cycles. Thenumber of times and the frequency that the valves 242, 243 open andclose are controlled and monitored by apparatus software and can be usedto determine the amount of hydrogen peroxide solution removed from thereservoir and to calculate the theoretically remaining amount ofsolution in the reservoir, based on the total amount aspirated from thesupply bottle and the metered amount. The inventors of the presentapparatus and method have discovered that, contrary to common generalknowledge the exact amount of evaporated hydrogen peroxide supplied intothe chamber is not critical. To the contrary, the inventors of thepresent application have surprisingly discovered that the most reliabledeterminant of the sterilization efficacy of the hydrogen peroxide vaporis the pressure in the chamber. The sterilization efficacy is dependenton the saturation level of the sterilization atmosphere with hydrogenperoxide. However, the saturation level cannot be calculated reliablyfrom the amount of solution injected, since it greatly depends on theload in the chamber and the adsorption characteristics of the materialsin the load. The saturation level is however directly proportional tothe pressure in the chamber. Therefore, the saturation level in thechamber can be determined solely on the basis of the chamber pressurerather than by measuring the flow or amount of the injected hydrogenperoxide solution into the chamber. As a result, the number of valveswitching cycles during the hydrogen peroxide injection step 360 in anembodiment of the present invention is wholly dependent on the pressureto be reached in the chamber 10 at completion of the hydrogen peroxideinjection. In a preferred embodiment, a 50% aqueous hydrogen peroxidesolution is used and the pressure increase to be reached in the chamberis 19 Torr. An optional dwell time of 2 minutes follows the reaching ofthe preset pressure increase of 19 Torr. Then a dose of dry ozonecontaining gas is injected followed by a second exposure time. The ozonedose depends of the cycle chosen by the user. When the desired number ofrepetitions of the first and second partial sterilization steps isattained, ventilation of the sterilization chamber 10 is carried out byevacuating and re-filling the chamber 3 times with oxygen in order toremove residuals of the hydrogen peroxide and ozone sterilants.

In order to determine whether a variation in the volume of hydrogenperoxide injected by each pulse during the conditioning phase has animpact on the sterilization effectiveness and on the amount ofcondensation observed on the load, applicant performed sterilizationtesting with different injection pulse amounts. Theoretically, the speedof injection/evaporation of the hydrogen peroxide could have an impacton the sterilization effectiveness. By injecting a much larger volumeduring each pulse, the solution is pushed faster into the chamber, andthe time for the liquid to evaporate is diminished. The chance of havingmore condensation on the instrument or on the packaging material istherefore greater. Condensation that is too pronounced would be expectedto create two problems. First, pronounced condensation could limit theability of ozone to reach the spores at the surface of the instruments.Second, the hydrogen peroxide liquid can stay trapped in the packagingmaterial, being hazardous for people handling the sterilized loadafterwards. If the amount of trapped hydrogen peroxide liquid is toolarge, ventilation of the chamber and packaging at the end of thesterilisation cycle may not be sufficient, to remove all traces ofhydrogen peroxide condensate.

When the pressure in the sterilisation chamber is lowered belowatmospheric pressure, any liquid present or injected into the chamberwill boil at a lower temperature than at atmospheric conditions. In theabove described embodiment of the present process, the pressure in thechamber is first lowered and then a volume of hydrogen peroxide isinjected in vapour form. The total volume of hydrogen peroxide used isinjected in small increments. During injection, the pressure in thechamber increases until a final pressure of 20 Torr (1 Torr startingpressure +19 Torr pressure increase) is reached. Hydrogen peroxideevaporates at a temperature higher than water (50% hydrogen peroxideboiling point is 114° C., and water boiling point is 100° C.).Therefore, the condensate will be more concentrated in hydrogen peroxidethan the initial solution entering the chamber. This phenomenon wasobserved with a UV lamp placed in the chamber. Even if the pressure inthe chamber was increasing, the concentration of hydrogen peroxide invapour read by the UV lamp was decreasing. Also, the concentration ofthe first hydrogen peroxide droplet (10 Torr) was titrated. It was foundthat the liquid was approximately 85% concentrated hydrogen peroxide.However, condensation of the hydrogen peroxide interferes with thesterilization of long lumens, since the hydrogen peroxide is removedfrom the vapor phase during condensation. Thus, in order for thehydrogen peroxide to penetrate long lumens, the hydrogen peroxide shouldbe maintained in the vapor phase as long as possible and condensationavoided during hydrogen peroxide injection.

At a pressure of about 10 Torr, a layer of micro-condensation of thehydrogen peroxide appeared on objects in the chamber. The thickness ofthe micro-condensation was calculated to be only a few molecules thick,but can assist the sterilisation, since it is well known that hydrogenperoxide can sterilize in a vapour form as well as in liquid form (Chunget al., 2006; Unger-Bimczok et al., 2008). Also, ozone is more solublein hydrogen peroxide and can form radicals right at the surface, wherespores are present.

In order to inject a high volume at once, a valve separated by Teflontubing was used instead of the normally used microvalve (AM-213-001,TSO3 Inc.). The tubing length was determined by the volume to beinjected. Since the volume contained in the valve is significant, twosizes of valves were used. The first type (TSO3#: CM-900-157) with anorifice of 0.062″, was used for a volume up to 1.5 mL. The secondNeptune type, with an orifice of 0.156″, (CM-900-156, TSO3 Inc.), wasused for a volume up to 3.5 mL. The larger valve size also helps to pushthe large liquid volume into the chamber. For the 35 μL volume, a Burket7616 micropump (CM-113-001, TSO3 Inc.) was used. For the 23 μL volume, alarger, specially-made block was used.

Two cycles were used for this experiment. To test the sterility, Cycle 1(half-cycle) was used, where the injection step of the conditioningphase was modified with a variation in volume and pulse for eachattempt, as previously described. As for the condensation effect, Cycle3, consisting of four phases, was utilized. This cycle was chosen due tothe fact that a greater quantity of hydrogen peroxide was injected forthe cycle, making it the worst case scenario. A third test was performedfor sterility testing. Lumens (Teflon 1 mm×80 cm) were inoculated usingthe wire technique according to MCB-09-A07. After exposure to ahalf-cycle of Cycle 1, the sterility of each lumen was determinedaccording to MCB-09-A04 rev.7 by quantitative recovery using theultrasound technique followed by filtration.

A burette was plugged onto the valve system in order to preciselydetermine the injected volume. This volume was then divided by the pulsenumber. The three TSO3 cycles were tested with a special loadrepresenting an average load for these three cycles. The load was alwaysat room temperature at the beginning of the cycle. A UV lamp was alsoinstalled on the sterilizer used. This allowed analysis of the hydrogenperoxide vapour during the conditioning phase

Sterility was verified with Teflon wires (1 mm×80 cm) inserted into thetubing, and tested in a half-cycle of Cycle 1. The first injected volumeby each pulse during the conditioning phase was 1.5 mL. In the case of agood result for sterile efficacy, the volume would be doubled. If theresult was not satisfactory, then half the volume would be tested. Sincethe result for the test using 1.5 mL per pulse was good, the test wasrepeated with 2.5 mL and 3.4 mL. Testing was stopped at 3.4 mL injectionbecause only two pulses were necessary to reach the desired pressure of18 Torr. The normal conditioning phase stopped at 19 Torr, but to ensurethe pressure was not exceeded, the microvalve was used between 18 to 19Torr.

Sterility was achieved with 3.4 mL (all tests were at zero for sporecount). Thus, applicant found that variations in pulse volume have noeffect on sterilization efficacy. However, it was noticed during thesterility testing that condensation was present exactly where thehydrogen peroxide is injected into the chamber. Therefore, more testswere performed in order to determine the maximum volume that could beinjected by each pulse without condensation.

The first volume injected was again 1.5 mL. Condensation was present onthe load at the injection site. The amount of liquid condensate measuredwas similar to that observed with a 3.4 mL injection pulse. The pulseamount was then gradually decreased by reducing the injected amount byhalf each time until no more condensation was visible. At 75 μL,condensation was again similar to that with an injection pulse of 3.4mL. A significant reduction in condensation build up was observed belowa pulse volume of 75 μL. At 35 μL, condensation was still visible butmuch reduced. At 23 μL, almost no condensation was visible. At a pulsevolume of 16 μL absolutely no condensation was observed. Condensationwas found to occur at pulse volumes above 20 μL. Thus, to control theamount of unwanted condensation of hydrogen peroxide, it is preferred touse a pulse injection volume of less than 75 μL, more preferably below35 μL, most preferably about 20 μL.

In an exemplary process in accordance with this disclosure, thesterilization chamber walls are maintained at a temperature of 40° C.while the load temperature may vary between 20° C. and 25° C. Theconcentration of the hydrogen peroxide solution used is preferably 50%,but, concentrations as low as 3% and as high as 59% can be used. Thepressure reached inside the chamber is a function of the hydrogenperoxide concentration used (see Table II). Even though the pressurereached is the same for each cycle discussed above, the volume ofhydrogen peroxide solution required depends on the concentration of thesolution, the type of load in the chamber and the hydrogen peroxideadsorption capacity of the load. The humidification level in thesterilization atmosphere prior to ozone injection can be adjusted byusing different concentrations of the hydrogen peroxide solution.

The dose of ozone varies between 2 mg/l for cycle #1 and 10 mg/l forcycle #2 and its exposure time varies between 5 minutes for cycle #1 and10 minutes for cycle #3.

The amounts of ozone used in prior art sterilization processes employinghumidified ozone as the sterilization gas are generally about 85 mg/l.Using hydrogen peroxide for partial sterilization as well ashumidification of the load prior to ozone injection allows for asignificant reduction in the amount of ozone required for achievingsterilization (SAL 10⁻⁶) down to a dose between 2 mg/l and 10 mg/l,depending on the cycle chosen. This reduction is much higher than wouldbe expected from just the fact that hydrogen peroxide and ozone are usedin the same sterilization cycle.

Indeed the evaporated hydrogen peroxide solution injected into thechamber is not sufficient to achieve sterilization, although a 4 logreduction in spores has been observed. However, adding only a very minoramount of ozone in the range of 1-10 mg of ozone per liter ofsterilization atmosphere results in full and complete sterilization atthe level required under the Security Assurance Level standards of theFDA or world standards, such as ISO (SAL 10⁻⁶). Such completesterilization could not be achieved using only the injection ofevaporated hydrogen peroxide solution, independent of the amount ofhydrogen peroxide solution used and the concentration of the solution.Moreover, high concentrations of hydrogen peroxide reduce compatibilitywith some instruments. In addition, a longer dwelling time afterhydrogen peroxide injection, for example 3 minutes instead of 2 minutes,does not enhance sterilization efficacy. In fact the dwelling time afterhydrogen peroxide injection appears to have no effect on sterilizationefficacy. Yet, adding only the minor amount of ozone as discussed abovesurprisingly leads to complete sterilization.

During the evacuation step 350 (see FIG. 3), oxygen supply valves 21 and26, mixture supply valve 29 a, and mixture bypass valve 29 b are closedand the chamber drainage valve 44 is opened. The sterilization chamber10 is evacuated to a vacuum pressure of about 1 Torr (1.33 mbar). Oncethis pressure is reached, which is determined by way of a pressuresensor 13 on the sterilization chamber, the chamber drainage valve 44 isclosed and the metering unit 240 activated to supply hydrogen peroxidesolution to the evaporator unit 260 in which the solution is evaporatedand subsequently flows freely into the sterilization chamber 10. Once apressure increase of 19 Torr is reached in the sterilization chamber 10,as determined by pressure sensor 13, the metering unit 240 isdeactivated and the supply of hydrogen peroxide solution to theevaporator 260 is stopped. The chamber can be maintained sealed so thatno injection of any substance occurs during a following first exposureperiod 370, which may lasts for 2 minutes. However, that exposure periodis completely optional. Shortly before the end of the hydrogen peroxideinjection step 360, (usually about 2 to 6 min.), the ozone generator isactivated to ensure a supply of ozone containing gas. The flow of theoxygen/ozone mixture exiting the ozone generator is controlled at alltimes by regulator orifice 28 capable of resisting the vacuum and ofadjusting the flow to between 1 and 3 litres per minute. Activation ofthe ozone generator 22 includes opening of supply valve 26 and mixturebypass valve 29 b. Supply valve 26 lets oxygen enter the generator. Theozone-oxygen mixture produced by the generator is then guided directlyinto the ozone catalyst 52 through mixture bypass valve 29 b. Aftercompletion of step 370, the oxygen-ozone mixture produced by thegenerator 22 is guided into the sterilization chamber 10 by opening themixture supply valve 29 a and closing the mixture bypass valve 29 b. Theoxygen-ozone mixture enters the chamber 10 until the desired ozoneconcentration according to the cycle chosen is reached in the chamber.The time required for this step is dependent on the flow rate andconcentration of the ozone gas in the mixture (preferably 160 to 200mg/l NTP), as determined by an ozone monitor 15 of a type well known inthe art. Once the desired concentration is reached, the mixture supplyvalve 29 a is closed to seal off the sterilization chamber and tomaintain the ozone/oxygen gas mixture in the chamber under vacuum.

Once the supply of the sterilization gas (mixture of oxygen and ozonegas) into the chamber is stopped, the generator 22 is stopped and theoxygen supply valve 26 is closed. The chamber is maintained sealed foran exposure period of 5 to 10 minutes, depending on the sterilizationcycle chosen by the user. Also dependent on the cycle chosen, steps 350to 390 are repeated 1 to 3 more times before the sterilization iscomplete. This set-up conformed to the Security Assurance Levelstandards of 10⁻⁶ (SAL 10⁻⁶).

To remove all remaining hydrogen peroxide, ozone and humidity in thesterilization chamber 10 after complete sterilization, the ventilationphase 400 is engaged. The ventilation phase begins after the lastexposure period 390. The chamber drainage valve 44 is opened and avacuum is applied down to approximately 6.5 mbar. Once the vacuumpressure of 6.5 mbar is obtained, drainage valve 44 closes and theoxygen supply valve 21 opens, admitting oxygen into the sterilizationchamber 10. Once atmospheric pressure is reached, the oxygen supplyvalve 21 is closed, the sterilization chamber drainage valve 44 isopened, and vacuum reapplied until a pressure of 1.3 mbar is reached.This last ventilation cycle, down to 1.3 mbar, is repeated once for atotal of three ventilation cycles. Once atmospheric pressure is reachedafter the last cycle, the door mechanism of the sterilization chamber isactivated in step 410 to permit access to the contents of thesterilization chamber. The ventilation phase has two functions. First,to remove all sterilant residues in the sterilization chamber beforeopening the access door and, second, to dry the sterilized material byevaporation when the vacuum pressure is applied. Of course, differentvacuum pressures, cycle times and number of repetitions can be used, aslong as the desired sterilant removal and drying are achieved.

The sterilants and humidity containing gas evacuated from thesterilization chamber 10 is passed over the catalyst 52 prior toexhausting the gas to the atmosphere to ensure a complete decompositionof the sterilants. The catalyst 52 is used during only two portions ofthe sterilization cycle, the activation of the generator 22 (with valves26 and 29 b) and the evacuation of the sterilization chamber 10. Duringthe start up phase of the generator 22, the mixture bypass valve 29 b isopened and the ozone is guided across the catalyst 52. Once the start-upphase of the generator 22 is complete, the bypass valve 29 b closes.During ventilation of the sterilization chamber 10, the sterilizationchamber drainage valve 44 is opened and the ozone containingsterilization waste gas is guided to the catalyst 52. Once theevacuation of the sterilization chamber 10 is completed, the drainagevalve 44 is closed. The circulation of ozone is ensured by the vacuumpump 40. The catalyst 52 can be located upstream or downstream of thevacuum pump 40.

In effect, at 20° C., water boils up to an absolute pressure of 23,3mbar and at 35° C., water boils up to an absolute pressure of 56,3 mbar.The vacuum in the sterilization chamber is preferably adjusted at apressure where the boiling temperature of water is lowered below thetemperature in the sterilization chamber. That boiling temperature maybe so low that the temperature of the hydrogen peroxide solution in theevaporator unit would decrease rapidly and, depending on the energyavailable from the surrounding structure, may freeze if no energy supplyis provided. The energy needed to evaporate the hydrogen peroxidesolution is taken from many sources. It is taken principally from themain body of the evaporator unit 260, which is in the form of analuminum block provided with a heating arrangement (not shown). Theevaporation process may also cool the humidifier to a point wheremoisture condenses on the sterilization chamber walls. This is avoidedby heating the chamber walls sufficiently to keep them at least at roomtemperature, preferably at 40° C. This is achieved with a heatingarrangement (not illustrated), which will be readily apparent to theperson of skill in the art.

The evaporated hydrogen peroxide solution injected into the chamberincreases the relative humidity in the sterilization chamber. Thishumidification significantly improves the efficacy of the ozonesterilization step. Oxygen/ozone-containing sterilization gas isinjected into the humidified sterilization chamber at a temperatureclose to ambient. The ozone-containing gas is not heated prior toinjection.

Hydrogen peroxide has its limitations when it comes to sterilizingmedical instruments. H2O2 is less stable when in contact with metal, asfor example, stainless steel. This problem is aggravated at lowpressures, at which chemical reactions are accelerated. Therefore, thedecomposition of hydrogen peroxide will be accelerated under vacuum,limiting the time available to sterilize long metal tubing. Moreover,the diffusion of H2O2 is limited since it is not a gas. Hydrogenperoxide would reach the end of long tubing by way of diffusion, but bythat time its concentration will have decreased, due to accelerateddecomposition, to a level where it is no longer sufficient forsterilization.

Applicants have discovered, as disclosed above, that these problems cannot only be overcome by the addition of a sterilant gas such as ozone,but that the humidification of the chamber by decomposition of thehydrogen peroxide into free radicals improves the efficacy of thesterilant gas. Moreover, applicants have surprisingly discovered thatozone can be advantageously replaced by nitrogen monoxide, or nitricoxide. The applicants discovered that the water and oxygen generatedduring hydrogen peroxide decomposition also improves the efficacy of thenitric oxide.

Nitrogen monoxide (or nitric oxide) is known to be cell toxic at lowconcentrations. In the presence of water and oxygen, NO reacts to formnitrogen dioxide, NO2, which is also highly toxic. In the absence ofoxygen, NO does not form NO2, but reacts to form nitric acid, which isvery corrosive to other materials.2NO+3 H2O2→2HNO3+2H2O   (1)2NO2+H2O2→2HNO3   (2)

The problem of nitric acid formation is minimized by mixing the nitricoxide with hydrogen peroxide instead of water, since the required NOconcentration after hydrogen peroxide pre-conditioning is very low. H2O2treatment, weakens the spore coat, and hydrogen peroxide and nitricoxide, when mixed together, form free radicals, similar to the reactionof ozone when mixed with hydrogen peroxide.HO+H2O2→H2O+HO2.   (3)HO2.+NO→HO.+NO2   (4)HO.+NO→HONO   (5)

Those radicals will react rapidly with all organic substances, oxidizingthem. The speed of oxidation will be in the order of 109, instead of 101for NO or O3 alone.

Applicants tested the efficacy of replacing the ozone gas originallytested by another gas, such as oxygen and nitric oxide. The testevaluated the sterile efficacy on inoculated devices. Inoculated wireswere inserted in tubing and afterwards in pouches. The pouches were alsoplaced at the top of the loading carriage in the sterilization chamber.This area is considered the point of least efficacy in the chamber.

EXAMPLES

The same loads were used for the three series of tests performed: ozone,oxygen and nitric oxide. The length, diameter, material and type oftubing were different for each cycle and are described in Table 3. Theinoculated lumens were placed in a special load representing an averageload for the three cycles.

TABLE 3 Length, diameter and material of tubing for each cycle. Cyclenumber Diameter (mm) Length (cm) Material Cycle 1 1 80 Teflon Cycle 2 150 Stainless steel Cycle 3 1 110 Teflon

The lumens used to evaluate the sterile efficacy were inoculatedaccording to protocol MCB-09-A07 rev 9. The wire method was used. Thewires were inoculated with 10 μL of a G. stearothermophilus ATCC 7953spores suspension of 1.0×10⁶ to 2.5×10⁶ UFC/10 μL. The inoculated wireswere left to dry overnight at normal room conditions.

Test loads were exposed to a half-cycle of each cycle. For theexperiment with oxygen and nitrogen oxide, ozone was replaced by the gasto be tested. A burette was also plugged on the valve system in order toprecisely determine the H2O2 injected volume. After the exposure, thesterility of each lumen was determined according to MCB-09-A04 rev.7 byquantitative recovery using the ultrasound technique followed byfiltration.

Ozone

The baseline of sterile efficacy on the inoculated lumens used in eachcycle was established using only hydrogen peroxide. Cycles usinghydrogen peroxide and ozone were performed to compare the efficacy ofoxygen and nitrogen oxide to ozone.

Oxygen

The oxygen was injected in the chamber using the same system as thatused for ozone. The ozone generator was turned off.

Nitric oxide

The NO was injected however directly in the chamber from an independentNO cylinder (Praxair). A Neptune valve with an orifice of 0.156″(CM-900-156, TSO3 Inc.), separated by a Teflon tube was used for thisinjection. By doing so, the gas was forced into the chamber.

All tests were performed outside in order to limit possible dangers fromaccidental leaks. A NO detector was used. A long tube was plugged intothe catalyst converter unit, to allow the NO to be eliminated far fromthe set-up. A calculation was performed (see below) to determine thenumber of valve injections necessary to obtain a concentration of 2mg/L.

Valve volume: 3.3 mL (Volume calculated in R-1937)

NO Density NTP: 1.25 g/L

Sterilisation chamber volume: 125 L

Finale concentration desired: 2 mg/L

NO Pressure: 3 psig

Corrected volume: 3300×((14.7 +3)/14.7)=3973.2 μL

Mass to be injected: 0.002 g/L×125L=0.25 gno

Masse injected by each injection: 1.25 g/L×0.003974 L=4.9665×10-3g/injection

Number of injections required: 0.25 gno /4.9665×10-3 g/injection=50injections

Two lenses were present in the chamber, one at the bottom rear, and theother one at the top rear. They were exactly aligned one on top of theother. One lense emitted UV light from a tungsten source, and the otherlense was connected to a UV detector. This set-up allowed themeasurement of the hydrogen peroxide vapour in the chamber.

Hydrogen peroxide has some inactivation activity against spores of G.Stearothermophilus. However, the percentage of sterility achieved inlumens is not sufficient to use it alone, especially for rigid and longflexible lumens. Results for hydrogen peroxide and of other gases mixedwith the hydrogen peroxide are summarized in Table 4.

TABLE 4 Percentage of sterility for the three TSO₃ cycle with differentsterilizing agent mixed with hydrogen peroxide. Sterilizing AgentSterile lumens Used Cycle 1 Cycle 2 Cycle 3 H₂O₂ 50% 12.5%  16% H₂O₂ +O₃ 77% 50% 77% H₂O₂ + O₂ 11%  0% 77% H₂O₂ + NO 100%  66% 66%

In the case of oxygen mixed with hydrogen peroxide, concentrationsequivalent to the ozone dose were used in each cycle, in other words, 2mg of O2/L for cycle 1, 10 mg/L for cycle 2, and finally 3 mg/L forcycle 3. Oxygen hindered the efficacy of the process in Cycles 1 and 2compared to hydrogen peroxide alone or mixed with ozone. In Cycle 3, theefficacy of the process with oxygen or ozone is equivalent.Consequently, oxygen was found ineffective to replace ozone.

Although nitric oxide is a well known disinfecting agent, it was nevermixed with hydrogen peroxide, since the mixture can be explosive at highconcentrations. To minimize the explosion danger, the NO concentrationwas limited to 2 mg/L for three cycles of a first series of tests.Sterility was achieved for some samples in all of the cycles so thenitrogen monoxide concentration was not further increased. The resultswere very conclusive, i.e., better than or similar to ozone mixed withhydrogen peroxide.

Even if no controls were done to verify the inactivation of G.stearothermophilus spores by NO in this study, it was demonstrated inmultiple studies that the inactivation rate of NO is low. When NO isinjected into a sterilization chamber and combined with humid air, theNO reacts with the oxygen at a predictable rate to form NO2, which islethal to the spores of G. stearothermophilus. When NO is injected intoa sterilization chamber with no oxygen atoms present, the NO does notform NO2, and spores are not sterilized(http://vvww.mddionline.com/article/sterilizing-combination-products-using-oxides-nitrogen).Based on the Noxilizer sterilization process publisher data, at 5.12mg/L NO2, the D-value is only 0.3 minutes. At 3 mg/L, the D value isapproximately 1.9 minutes.

In this experiment, the amount of NO injected was 2 mg/L. Consideringthat all NO molecules were transformed in NO2, a D-value of 1.9 minutesfor a concentration of 2 mg/L of NO2, only 2.5 log of spores would havebeen inactivated by the NO2. This less than the 6 log present on theinoculated devices. In reality, the conversion rate of NO in NO2 isprobably not 100%, and the D-value is more than 1.9 minutes. Thus thenumber of spores inactivated by NO only is probably more around 1 log.

The substitution of ozone by another gas was tested in all three cyclesof the present process. Hydrogen peroxide injection was performed asusual. Two gases were tested. The first, oxygen, did not achieveconclusive results. Sterility was not achieved in two of the threecycles.

Nitric oxide was also tested. Results show a complete sterility in allthree cycles. The concentration used for all tests was low. Only 2 mg/Lwas injected for the three tests. The use of this chemical could beconsidered in the future. However, significant changes to the sterilizerwill have to be made to accommodate this. Since NO₂ is formed during thecycles, only compatible materials could be used Also, protectiveequipment, like for example NO detector would have to be considered.

Other sterilant gases that can interact with hydrogen peroxide tocontinue the formation of free radicals could be used in replacement ofozone, such a chloride dioxide.

On the other hand, many different molecules can have the same effect ashydrogen peroxide on ozone. Some ions can also have the catalytic effectof hydrogen peroxide on ozone. Co²⁺, Ni²⁺, Cu²⁺, Mn²⁺, Zn²⁺, Cr²⁺ andFe²⁺, Ti²⁺ ions enhance the decomposition of ozone (Ahmed et al., 2005).All transition metals that can form a molecule with oxygen willdecompose ozone. The positive ions will try to become neutral by takingan oxygen atom to the ozone molecule. The ozone molecule being more orless stable will easily give the oxygen atom. Water with a basic pH willbe richer in hydroxyl ions. Hydroxyl ions decompose ozone into atomicoxygen. Those oxygen atoms can form hydroxyl radicals afterward.Therefore, any molecules that can be used to render the solution pHbasic will favour the decomposition of ozone. Good examples are NaOH orKOH.

Another source of hydroxyl radicals are all solvents containing analcohol group. Those solvents will provide OH ions and will favour thedilution of ozone. In the same vein, formate and humic substances caninitiate the chain towards radical formation (Glaze et al., 1987). Someacids can also be used such as acetic acid and para-acetic acid. Ozonebeing more soluble and stable in acidic solution will be able to reactlonger and be more concentrated. Any molecule containing a carbonate,bromine, phosphate or sulphate group will also decompose ozone (Beltrán,2004).

As shown in FIGS. 2 and 7, the delivery unit 200 includes a bottleholder 202 for receiving a sealed hydrogen peroxide solution bottle 180.The holder has a bottle seat 204 in which the bottle 180 is fittinglyreceived. The bottle 180, which will be discussed in more detail furtherbelow, is held in the seat 204 by gravity only. The holder 202 isrotatably mounted on pivot 203 for movement between an open position asillustrated in FIG. 7, which the bottle 180 can be placed into orremoved from the holder and a closed position in which the holder iscompletely within the sterilizer cabinet (not shown) and a front cover205 of the holder closes off all access to the holder from outside thecabinet. When the holder 202 is in the closed position, a pneumaticallydriven drainage arrangement 207, including a needle drive, in thisembodiment a vertically oriented pneumatic cylinder 208, and a drainageneedle 209 mounted on the piston rod 210 of the cylinder, is activatedto drain all hydrogen peroxide solution from the bottle 180. This isachieved by activating the cylinder 208 to force needle 209 through thebottle seal until the needle tip reaches the bottom of the bottle 180.The needle 209 is fluidically connected to the reservoir 240 (see FIG.8) and the solution is aspirated from the bottle 180 and into reservoir240 by using the vacuum generated by the vacuum pump 44 to which thereservoir 240 can be fluidically connected by conduit 211 and valve 212(see FIG. 1). Once the contents of the bottle 180 have been aspirated,the holder can be opened and the bottle removed, or the empty bottle canbe kept in the holder until a refill of the reservoir 240 is required.The reservoir 240 is provided with a level sensor 242 which provides asignal to the control system on the liquid level in the reservoir. Basedon the signal received from the sensor 242, the control system notifiesthe user if the amount of liquid in the reservoir 240 is insufficientfor the execution of the cycle selected by the user.

In an alternate embodiment, the hydrogen peroxide delivery system doesnot include a reservoir. Instead, the bottle 180 itself is cooled down(CS-01) to avoid rapid degradation of the aqueous hydrogen peroxide. Asensor (S14) measures the amount of solution left in the bottle. Whenthe solution reaches a 1^(st) preselected level, a 1^(st) warningappears on the screen and when a lower, 2^(nd) preselected level isreached, the message generated from the software to the operatorspecifies that only one more sterilization cycle #1 or #2 can be runwith the remaining solution in the bottle. The operator will then haveto reload the delivery system with a fresh, full bottle.

As shown in FIGS. 10a to 10d , the bottle 180 has a conical bottom 182to ensure a complete drainage of all liquid in the bottle, therebyreducing the danger of spills or contamination on removal of a drainedbottle. In order to ensure the bottle 180 securely remains upright, astand 184 is attached to the bottom end of the bottle. The stand 184includes an upturned cup 185 snap fitted into a circumferential groove186 on the bottle exterior wall 187. The needle 209 is aligned with thelowest point in the bottle bottom and can be moved into the bottle,through the bottle seal, until it reaches the lowest point in thebottle. Mechanical, electronic or other control structures and functionsare provided to ensure contact of the needle with the bottle bottomwhile preventing penetration of the bottle bottom. A pressure sensor ispreferably incorporated into the reciprocating needle drive and/or theneedle mount (not shown).

Control System

The sterilization apparatus is preferably controlled by the schemepresented in the electrical block diagram (FIG. 9 and Process FlowDiagram (FIG. 3). The control system is built around a PLC shelf(Programmable Logic Controller). This shelf contains a power supply(107) a CPU unit (108), a Device Net Transceiver (109), a 32×24 Volt DCdiscrete input module (110), a 16×120 VAC discrete output module (111)and finally 16 transistor discrete output module (112), an RS232Ccommunication module. All those modules are stacked together by anintrinsic connecting system that contains a data and address bus.

Device Net is an industrial serial communication protocol largely usedin the industry for instrumentation and control. In this sterilizationapparatus, the Device Net transceiver (109) is used to communicate infull duplex, the data between the CPU (109) and the 15 bit A/D converter(106), a 15 bit D/A converter (125) and both Digital TemperatureInterfaces (120), (121).

The PLC CPU possesses three RS232 ports. One is used to receive and senddata to the Touch Screen Terminal (118), another one is used to senddata to a thermal printer (119) and the last port is used as a serviceport where a PC (Personal Computer) can be hooked up to communicate withthe PLC CPU (108) to load up the control protocol program. (ControlProtocol Program is not in the scope of this document).

The Touch Screen terminal (118) is located at the front of thesterilizer beside the thermal printer (119). Touch Screen Terminal andthermal printer constitute a User Interface terminal.

Power needed for: thermal printer (119), Device Net Link, (109), (106),(120), (121), (125), Chamber Pressure Sensor (104) electronic oxygenregulator (126) and PLC discrete inputs (111) and discrete outputs (112)is provided by the DC Power Supply (103).

Chamber Pressure Sensor (104) and Ozone Monitor (105) have a standard 0to 10 VDC output signal. Electronic Oxygen Regulator have an output of 0to 5 VDC. All signals are sent to a 15 bits A/D converter. All convertedsignals are sent to the CPU by the Device net digital link forprocessing.

Power input (100) of the sterilizer is a three wire 208 to 240 VACsingle phase type without neutral. The power input is filtered toprevent conducted RFI (101). The power is distributed by powerdistribution buss (102) to the various electrical systems of thesterilizer apparatus.

A cooling system (60) is used to cool down the ozone generator. Thissystem includes the cooling unit (114) and the coolant circulator pump(113). The temperature of the coolant in the generator is sensed by anRTD located at the generator. The temperature is sent to the CPU (108)by the Device Net system (109) (120) (121). Coolant circulator (113) andcooling unit (114) are controlled by contactors driven by PLC outputs(111) which in turn are controlled by the software protocol. All inputand output required to achieve cooling system control are listed on theelectrical block diagram as: Circulator Pump Relay, Cooling SystemRelay, Circulator Overload Sensor, Cooling System Overload system,Refrigerant Low Pressure and Coolant Flow Switch.

The vacuum control system includes the vacuum pump 40 and a pressuresensor 104. The start and stop operations of the vacuum pump arecontrolled according to the control protocol. All input and outputrequired for the vacuum system is listed on the diagram: Vacuum PumpContactor, Vacuum Pump not running sensor, Vacuum pump Overload sensor,Vacuum to Chamber Valve (44), Air Pulse Valve (18) and Oxygen to ChamberValve (21). The pressure sensor output is converted by the 15 bit A/Dconverter (106) and sent to the CPU by the Device Net digital Link(109). The pressure sensor also possesses two discrete outputsindicating to the CPU (108) the following conditions: Chamber PressureSensor at Temperature and Chamber Pressure Sensor Heater failure. Thosetwo signals are listed on the electrical block diagram as PLC inputs.

The sterilization chamber door actuator system includes an electricdrive of the screw type and four inductive sensors which allow thedetection of the closure of the door and the locked or unlocked positionof the actuator as part of the control protocol. The door opening systemis also used in the alarm conditions management protocol to assure thesafety of the user. All input and output required to achieve the dooractuator system are listed on the electrical block diagram as: Lock DoorRelay, Unlock Door Relay, Door closed Lower Sensor (S2), Door closedUpper Sensor (S1), Door Locked Sensor (S4) and Door Unlocked sensor(S3).

The Ozone power supply (116) includes a full wave rectifier, anoscillator circuit and a high voltage transformer. The output of thetransformer is hooked up to the ozone generator (22). The power supply(116) is mounted as a resonator using the non-ideal characteristics ofthe high voltage transformer. The CPU 108 controls the ozone productionand ensures by way of the ozone monitor 104 and Electronic oxygenregulator (126), that the concentration desired for sterilization isachieved and maintained throughout the sterilization cycle. All inputand output required by the Ozone Generation System is listed on thediagram as: Oxygen Supply Valve (26), Ozone to Chamber Valve (29 a),Ozone Dump to Catalyst Valve (29 b), Ozone Monitor Zeroing), HighVoltage Standby Relay, High Voltage Current Limiter, Ozone High VoltageOverload sensor Rectifier High Temperature Sensor, Ozone monitorFailure.

The oxygen supply system is a unit called Electronic Oxygen PressureRegulator. A proportional Valve (26) which also shuts off the oxygen iscontrolled by an integrated PID circuit converting an analog signal froman absolute pressure sensor (27). Then the PID sends the appropriateduty cycle current to the proportional valve (26). With the orifice 28this system constitutes an oxygen flow regulator. The mechanicalregulator 24 is used as a first stage regulator to lower the oxygenpressure of 60 psi to 10 psi. The electronic regulator also provides thealarm condition protocol to ensure the protection of the user. Inputsused for the alarm condition are listed on the electrical block diagramas: Oxygen High Pressure Sensor and Oxygen Low Pressure Sensor. Also,the electronic oxygen pressure regulator provided a 0 to 5 VDC analogoutput read by the A/D converter 106 trough device net network.

The control system is provided with a user interface 118. In thepreferred embodiment, this interface includes a touch-sensitive liquidcrystal display (LCD) screen 118, a printer 119 for performance reportsand a communications port 153 (Series RS-232) allowing the user toreceive and transmit information necessary for use of the apparatus. Itwill be readily apparent to the person skilled in the art that othertypes of user interfaces can be used such as touch-sensitive pads,keyboards, or the like, and other types of communications interfaces.Thermal printer status inputs appear on the electrical block diagram as:Printer Off Line Sensor and Printer Out of Paper.

H2O2 Dispensing System Control Processing

At the moment, two configurations of an H2O2 dispensing system arepossible. The control system could be used for both systems. The firstsystem depicted in the present application in FIG. 7 and FIG. 8 ismainly a bottle of H2O2 (180) flushed into a temperature controlledreservoir (240) FIG. 8. This first system will be described withreference to FIGS. 7, 8, 9 and 2. All input and output sensors describedin the following appear in the list of inputs and outputs of the controlsystem listed on FIG. 9. When the sterilizer is first initialized, thedoor 12 is closed and the closed position is sensed by switch S7. Nobottle is sensed in the holder by (S6), the puncture needle is alsoretracted to the up position by the cylinder PA-01 (208). S8 and S9provide sensing for the upward and downward position of cylinder (208).Also, actuator PA-02 is retracted in the holder unlocked position. Theuser is invited by the message on the screen (118) to open the door(205) and to insert a H2O2 bottle in the holder. So when the bottle issensed by S6, another message on the screen (118) invites the user toclose the door (205) which is sensed by S7. Software control is carriedout by the CPU (108) and condition sensors. The bottle is set by gravityon a rotating base (209). The CPU starts the motor M-02 to rotate thebottle 180. A bar code reader BS-01 (FIG. 2,) (122) FIG. 9 reads a barcode on the bottle. The CPU verifies the expiry date of the bottle andif the bottle is past its expiry date, the door 205 remains unlocked anda message on the screen (118) invites the user to change the bottle foranother one. If the date is correct, the CPU stops the motor M-02 andlocks the door (205) by actuating PA-02 (FIG. 2). Then CPU actuates thecylinder (208) for the needle 209 to perforate the sealed cap of thebottle until S9 senses the needle in the down position. Then the bottleis totally emptied into the reservoir 240 by suction provided throughvalve (212) and vacuum from pump (40). The door (205) remains lockeduntil all the H2O2 in the reservoir has been used. Level sensors S10 andS11 provide the conditions necessary for the CPU to estimate if anotherbottle is needed. If so, the needle is retracted from the bottle and thedoor (205) is unlocked and the user is invited by a message on thescreen (118) to replace the H2O2 bottle.

Description of the Alternate and Preferred H2O2 Dispensing System

The following dispensing system does not include the cooled reservoir(240). Instead, the H2O2 remains in the bottle (180). Level detectorsS10 and S11 are removed and replaced by an ultrasonic level detectorwhich is spring loaded against a side of the bottle near the bottom andused as a low level detector to indicate to the CPU an empty bottle.Because this sensor is spring loaded, it adds too much friction on thebottle to use the motor M-02. Therefore, the user is invited by amessage on the screen (118) to rotate the bottle manually until the barcode is read by (BS-01) FIG. 2 or (122) FIG. 9. If the bottle is not outof date, the user is invited to close the door (205) and the CPU locksthe compartment of the bottle holder and actuates (208) to puncture downthe needle. In that preferred embodiment, the H2O2 holder is temperaturecontrolled by a Peltier cell unit. An RTD attached to the holder andconnected to the temperature interface (121) sends data to the CPU (108)by Device Net network and the CPU controls by PID function the amount ofpower being applied to the Peltier cell unit. The Peltier unit issupplied by the 12 VDC (121) power supply used also for the aircompressor driving the pneumatic system composed of SV-15, SV-16,actuators (PA-02 and PA-01) on FIG. 2 . Between each cycle, the lineconnected between the H2O2 bottle (180) and micro valve module (240)will be purged by SV20. Near the inlet of module (240) a foam opticaldetector snapped on the H2O2 line will indicate the total refill of theline without air in the line.

To that point both H2O2 dispensing systems can supply the micro valvesmodule (240). The micro valves (SV-18 and SV19) are working reciprocallyfor a preset duty cycle program on an on board microcontroller circuitgenerating the proper timing pulses for both micro-valves. Thatelectronic circuit is activated by a signal from the CPU (108) calledH2O2 pump controller signal FIG. 9. Under software control, a properamount of H2O2 is allowed in the humidifier manifold (260, FIG. 1). Thismanifold is temperature controlled by the CPU (108) using data of RTD(TT-04, FIG. 1) and controlling heater HTR-01 (FIG. 1) by PID function.Then the H2O2 vaporizes in the manifold (260) and the vapor is sent tothe chamber under vacuum through pipe (280, FIG. 1).

In the preceding description, for purposes of explanation, numerousdetails are set forth in order to provide a thorough understanding ofthe embodiments of this disclosure. However, it will be apparent to oneskilled in the art that these specific details are not required in orderto practice this disclosure. In other instances, well-known sterilizerstructures and circuits are shown in block diagram or symbol form inorder not to obscure this disclosure. For example, specific details arenot provided as to whether certain parts of the sterilizer controls areimplemented as a software routine, hardware circuit, firmware, or acombination thereof.

The above-described embodiments of this disclosure are intended to beexamples only. Alterations, modifications and variations can be effectedto the particular embodiments by those of skill in the art withoutdeparting from the scope of this disclosure, which is defined solely bythe claims appended hereto.

TABLE III Oxygen Circuit FTR-01 Oxygen Inlet Filter RG-01 OxygenPressure Regulator SV-01 Oxygen Supply Valve PS-01 Oxygen PressureSwitch FI-01 Oxygen Flow Indicator SV-05 Oxygen To Chamber Valve OzoneCircuit Ozone Generator TT-01 Temperature Transmitter for OzoneGenerator Cooling AOZ-01 Ozone Monitor Orifice (used to regulate ozoneflow to chamber) SV-02 Ozone To Chamber Valve SV-04 Ozone Dumped Valve(By-pass) Air Circuit AC-01 Air compressor AT-01 Compressed air tankPS-03 Pressure switch for air compressor RG-03 Air pressure regulatorPI-03 Air Pressure indicator FTR-03 Air inlet filter Aluminium BlockTT-04 Aluminium Block Temperature Transmitter HTR-01 Heating ElementSTERIZONE Solution Circuit SV-17 H₂O₂ filling valve SV-21 H₂O₂ ventvalve SV-18 H₂O₂ inlet valve SV-19 H₂O₂ outlet valve SV-20 H₂O₂ purgevalve STERIZONE Solution Supply System S6 Sensor (detects STERIZONESolution container presence- absence status) S7 Sensor (detectsSTERIZONE Solution compartment open- close status) S8 Sensor (detectsPA-01 upper position) S9 Sensor (detects PA-01 lower position) S12Sensor (detects STERIZONE Solution compartment locked- unlocked status)S13 Sensor (detects STERIZONE Solution compartment access (fascia)opened-closed status) S14 Sensor (detects the lower level of H₂O₂ in thebottle) S15 Sensor (detects presence of air bubble in the line) SV-15Air pilot valve for needle puncture actuators PM-900-014 SV-16 Air pilotvalve for STERIZONE Solution compartment lock actuator B-01 Custom tapershape bottom STERIZONE Solution bottle BS-01 Barcode scanner for bottlePA-01 Pneumatic actuator for bottle puncture PA-02 Pneumatic actuatorfor STERIZONE Solution compartment lock PA-03 Pneumatic actuator forpuncture needle centering M-02 Electric motor that rotate bottle forbarcode scanning CS-01 Cooling system for the bottle VS-02 Vacuum switch(to fill and purge H₂O₂ line) Sterilization Chamber S1 Door Closed UpperSwitch S2 Door Closed Lower Switch S4 Door Locked Switch S3 DoorUnlocked Switch PT-01 Chamber Pressure Transmitter VS-01 Chamber VacuumSwitch TT-03, Chamber Temperature Transmitters 5, 6 TT-07 Chamber DoorTemperature Transmitter Vacuum Circuit SV-06 Chamber Vacuum Valve M-01Vacuum Pump Run status flag M-01 Vacuum Pump Contactor CAT-01 CatalyticConverter Catalyst Drying Circuit FTR-02 Port muffler SV-11 Air toCatalytic Converter Valve (Catalyst Dryer Valve) PM-900-002 CoolingCircuit FS-02 Coolant Flow Switch M-05 Circulation Pump Run status flagM-05 Circulation Pump Contactor Overload Circulation Pump PS-02Compressor Low Pressure Switch M-06 Compressor Run status flag M-06Compressor Contactor Overload Compressor

The invention claimed is:
 1. A method of metering a preselected volumeof hydrogen peroxide, in a metering unit including a metering passagehaving a fixed known volume and extending through a body from anupstream end to a downstream end, the method comprising the steps of a)connecting the metering passage at the downstream end to a vessel undervacuum, while the upstream end is sealed, to evacuate the meteringpassage; b) sealing the evacuated metering passage at the downstreamend; c) while the downstream end is sealed, connecting the evacuatedmetering passage at the upstream end to a supply of hydrogen peroxidesolution for a time sufficient to draw the hydrogen peroxide solutioninto the evacuated metering passage and fill the metering passage withthe hydrogen peroxide solution; d) sealing the metering passage at theupstream end; and e) repeating steps a) to d); whereby steps a) to d)are repeated X number of times, X being a whole number represented by aquotient of the preselected volume divided by the fixed known volume andrepeating steps a) to d) leads to a flow of hydrogen peroxide throughthe metering passage from the upstream end to the downstream end.
 2. Themethod of claim 1, wherein the metering unit includes an intake valve atthe upstream end to seal the upstream end and an exhaust valve at thedownstream end to seal the downstream end and the valves are switchedoppositely so that one valve is closed while the other is open, toevacuate the passage when the intake valve is closed and the exhaustvalve is open, fill the passage when the exhaust valve is closed and theintake valve is open and once again evacuate the passage through theexhaust valve when the intake valve is closed and the exhaust valve isopen.
 3. The method of claim 1, wherein the known volume of the passageis between 75 μL and 15 μL.
 4. The method of claim 3, wherein the knownvolume is between 35 μL and 15 μL.
 5. The method of claim 3, wherein theknown volume is between 20 μL and 15 μL.
 6. A method of meteringhydrogen peroxide gas in a metering unit into an evacuated sterilizationchamber connected to an evaporator under vacuum, the metering unitincluding a metering passage having a fixed known volume and extendingthrough a body from an upstream end to a downstream end, the methodcomprising the steps of a)continuously monitoring a pressure in thesterilization chamber; b)connecting the metering passage at thedownstream end to the evaporator, while the upstream end is sealed, toevacuate the metering passage; c)sealing the evacuated metering passageat the downstream end; d) while the downstream end is sealed, connectingthe evacuated metering passage at the upstream end to a supply ofhydrogen peroxide solution for a time sufficient to draw the hydrogenperoxide solution into the evacuated metering passage and fill themetering passage with the hydrogen peroxide solution; e) sealing themetering passage at the upstream end; and f) repeating steps a) to d);until a preselected pressure is reached in the sterilization chamber;wherein the known volume of the metering passage is between 75 μL and 15μL.
 7. The method of claim 6, wherein the known volume is between 35 μLand 15 μL.
 8. The method of claim 7, wherein the known volume is between20 μL and 15 μL.